The biological evolution is based on the survival of specific genotypes that encode phenotypes with the most suitable functionalities in a certain environment. In all living species DNA programs the genotype. DNA serves two important functions in the natural selection process. One function is obviously to encode for the type of nucleotides used and the other function is to encode for the specific order of nucleotide sequences in a nucleic acid sequence. The strategy used in nature, i.e. encoding for the exact type as well as the precise sequence of nucleotides, ensures an extremely similarity between the progeny and its parents. Thus, conserving almost the exact sequence and type of the nucleotides is absolutely essential in order to create off spring with a high functionality. The changes in the genotype from one generation to another, which allow for evolution, are determined by the random mutation rate and recombination between the two parent's genotypes.
The natural selection cannot afford too many changes in the DNA from one generation to the next in order to secure survival of the species. Therefore, nature has evolved sophisticated means to proofread the copying of the DNA from the parents to its progeny and secured that the characteristics of phenotype from one generation to the next is carried only by the DNA.
Within the art of selecting ligands from a library of encoded polypeptides associated with a corresponding identifier nucleic acid sequence, the method of nature is used. Thus, when more than a single library generation is needed, the identifier nucleic acid sequences (genotype) carries the information from one generation to the next.
WO 93/03172 A1 discloses a method for identifying a polypeptide ligand having a desired property in a polypeptide library. In a first step, a translatable mRNA mixture is provided, which is mixed with a mixture of ribosome complexes to form a translation product attached to the mRNA strand responsible for the formation thereof. In a second step the ribosome complexes binding to a target are partitioned from and remainder of the library. In a third step, an amplification of mRNA strands of the partitioned ribosome complexes, which has bound to the target follows. The amplified mRNA strands are used for the production of a second generation library, which is subjected to a renewed contact with the target. The method is repeated a sufficient number of times until the size of the library has narrowed to a small pool of high affinity binders.
In WO98/31700 A1 a method for selecting a DNA molecule, which encodes for a desired protein, is disclosed. The method implies the initial presence of a pool of candidates RNA molecules, which subsequently is translated into a corresponding pool of RNA-protein fusions. Subsequently the mRNA-protein fusion products are subjected to a selection process, i.e. the fusion products are presented for a target molecule, and a new pool of complexes capable of binding to the target are partitioned. From the new pool of complexes, the mRNAs are recovered and amplified for use in a subsequent round of library generation. Xu, L. et al Chemistry & Biology, Vol. 9, 933-942, August 2002 discloses a practical embodiment in which a library of more than 1012 unique mRNA-protein fusion products through ten rounds of library generation and selection are used to identify a high affinity binding protein.
The preparation of libraries of synthetic molecules associated with a corresponding identifier nucleic acid sequence, and the selection of synthetic molecules from such libraries, have been the subject of various patent applications. When two or more generations of libraries are needed, the identifier nucleic acid sequence is used as carrier between an initial library and the next generation library.
Thus, in WO 00/23458A1 libraries of complexes comprising non-natural molecules attached to corresponding nucleic acid sequences are suggested. After a selection of the library has been conducted, the nucleic acid sequences of successful complexes are amplified by PCR and a new library is prepared from these nucleic acid sequences. The same method of carrying information from an initial library to the next library is applied in WO 02/074929A2 and WO 02/103008A2.
The present invention provides a new method for evolving encoded molecules. The method is based on the identification of chemical entities used in the synthesis of reaction products of successful complexes and the application, at least in part, of these chemical entities in the preparation of the next generation library. The utilization of preferable chemical entities and the exclusion of certain undesired chemical entities in the next library generation generally imply that the next generation library has a smaller size compared to the size of the initial library, thereby, at the same time, retaining the desirable encoded molecules in the library.